Brassica species are used as a source of vegetable oil, animal feeds, vegetables and condiments. Brassica plants that are used for vegetable production include cabbage, cauliflower, broccoli, kale, kohlrabi, leaf mustard and rutabaga. Seeds of B. hirta are used to produce the popular American condiment, yellow mustard. However, on a world-wide basis, the most economically important use of Brassica species is for the production of seed-derived, vegetable oils. The predominant Brassica species grown for oil production is B. napus, followed by B. juncea and B. rapa. Seeds of B. napus, B. juncea and B. rapa are referred to as rapeseed. Brassica species that are grown primarily for oil production are often called oilseed rape. In North America, canola, a type of oilseed rape that has been selected for low levels of erucic acid and glucosinolates in seeds, is the predominant Brassica plant grown for the production of vegetable oil for human consumption. While low-erucic-acid rapeseed oils, such as canola oil, may be favored for human consumption, high-erucic-acid rapeseed oils are desirable for a variety of industrial applications including the production of cosmetics, lubricants, plasticizers and surfactants.
Because of the agricultural and industrial importance of plants from the genus Brassica, plant breeders are working to develop new varieties with improved agronomic characteristics. While traditional breeding approaches are important, significant improvements in cultivated Brassica varieties have been made recently through the introduction of recombinant DNA into the Brassica genome by genetic transformation methods. A number of genetically modified Brassica varieties have already reached farmers' fields in North America. Transgenic canola varieties, genetically modified for resistance to herbicides, have rapidly gained favor with agricultural producers across the canola-growing regions of the United States and Canada. The phenomenal success of the transgenic canola varieties in North America has led to acceleration in the development of new transgenic varieties of canola. Novel, recombinant DNA-based strategies for incorporating new traits, such as disease and insect resistance, modified seed oil composition and modified seed protein composition, are being developed for canola and other Brassica species. All of these strategies depend on genetic transformation methods to introduce the recombinant DNA into the genomes of Brassica plants.
Currently, the most favored methods for transforming Brassica species involve the use of Agrobacterium. While the Agrobacterium-based transformation methods provide a reliable means for introducing foreign DNA into plants, there are a number of disadvantages to methods of plant transformation that involve the use of Agrobacterium. First, an undesired consequence of all Agrobacterium-based methods is the introduction of at least one T-DNA border into the genome of the recipient plant. While the T-DNA border is an essential element of the genetic mechanism by which Agrobacterium transfers DNA to a plant cell, the T-DNA border is not essential for the expression of foreign genes in the recipient plant. Additionally, the accumulation of multiple T-DNA borders throughout the genome of a plant may have deleterious effects on the plant or its progeny. Second, the co-cultivation of plant tissues with Agrobacterium may slow the regeneration of a transformed plant from a transformed cell. After the co-cultivation phase, Agrobacterium must be eliminated from cultures of the plant tissues. High levels of bactericidal agents must be applied to the plant cultures to kill the Agrobacterium. While the levels of bactericidal agents applied to the cultures are generally not lethal to the plant tissues, the presence of the bactericidal agents in the cultures may negatively impact plant growth and thus, slow the regeneration of transformed plants. Third, prior to DNA transfer to a plant, natural genetic processes might occur in Agrobacterium such as genetic recombination and DNA rearrangements that may have undesired effects on the DNA fragment that is transferred to the plant. Such undesired effects may alter or eliminate the intended genetic function of the introduced DNA fragment.
Efficient Brassica transformation methods that do not involve the use of Agrobacterium are desired. U.S. Pat. No. 6,051,756, U.S. Pat. No. 6,495,741, US 2003/0093840 and US 2004/0045056 describe the transformation of seedling hypocotyls by particle bombardment. U.S. Pat. No. 6,297,056 describes the transformation of cotyledonary petioles. U.S. Pat. No. 6,515,206 and US 2003/0200568 describe the use of transformation of plastids in true leaves. Chen and Beversdorf Theor. Appl. Genet. 88: 187-192 (1994) describe a biolistic transformation procedure of microspore-derived hypocotyls involving DNA imbibition. Fukuoka et al. Plant Cell Reports 17: 323-328 (1998) describe biolistic transformation of fresh microspores. Finally, Nehlin et al., Plant Physiol. Vol. 156: 175-183 (2000) describe transient biolistic transformation of pre-incubated microspores, but no stable transgenics were reported.
To meet the increasing demands of agriculture in the world today, the pace of development of new transgenic varieties of canola and other Brassica species must be accelerated. Increasing the pace of Brassica variety development depends on the availability of reliable and efficient methods for the transformation and regeneration of transformed Brassica plants.